Polypeptides and antibodies characteristic of papillomavirus, and diagnostic procedures and vaccines making use of them

ABSTRACT

The present invention provides papillomavirus polypeptides and antibodies against said polypeptides. The peptides and antibodies of the present invention are particularly useful for in the prevention, diagnosis, and treatment of infections caused by distinct papillomaviruses. These papillomaviruses are linked to distinct infectious states. The polypeptides of the present invention are derived from L2 genes of different papillomaviruses (or from a portion of said genes). The present invention further provides kits containing one or more antibodies according to the present invention, and a method for the detection and identification of papillomaviruses in biological samples by immunological reaction with said antibodies. The diagnostic kits of the present invention are suitable for diagnosis of the specific infection affecting the donor subject of the biological sample, or infections to which the subject risks being exposed.

This application is a divisional of application Ser. No. 08/426,648,filed Apr. 21, 1995 abandoned; which is a continuation of applicationSer. No. 08/232,588, filed Apr. 25, 1994 abandoned, which is acontinuation of application Ser. No. 07/999,583, filed Dec. 30, 1992abandoned; which is a continuation of application Ser. No. 07/693,088,filed Apr. 30, 1991 abandoned; which is a continuation of applicationSer. No. 07/507,007, filed Apr. 10, 1990 abandoned; which is acontinuation of application No. 07/289,452, filed Dec. 22, 1988abandoned, which is a continuation of application No. 07/050,904, filedApr. 22, 1987 abandoned which is a 371 of PCT/FR86/00288 filed Aug. 22,1986.

The invention relates to the DNAs of papillomaviruses, and moreparticularly to the probes derived from these papillomaviruses, as wellas procedures for their implementation in the in vitro diagnosis ofpapillomavirus infections.

The expression "papillomavirus" covers a large number of viruses, whichhave in common the property of being held responsible for several formsof viral infection extending from relatively benign epidermal andmucosal warts to hyperplasias liable to degenerate into intra-epithelialneoplasias and cutaneous cancers. Among these papillama infections,mention will also be made more especially of epidermodysplasiaverruciformis which will sometimes be designated below by the expression"EV".

A number of types of papillomavirus have already been described. Withinthe framework of the present patent application many new types andsubtypes of papillomavirus will be described which have been isolatedfrom warts or disseminated macular lesions, liable to give rise to thedevelopment of skin cancer in a high proportion of those patientsaffected.

Recent studies have revealed the importance of immune factors and themajor role played by various types of virus of human papillomas (HPV)factors which compound the role, already described in the literature,played by various genetic factors and actinic radiation in thepathogenesis of infections of papillomaviruses.

The invention results from observations made during the course ofcomparative studies of the behaviour of a large number of recentlyisolated papillomaviruses, the essential genomic characteristics ofwhich will be defined below.

The study of a small number of cases of EV had already led to thecharacterization of six types of HPV after molecular cloning of theirgenomes (KREMSDORF. et al, 1982, J. Virol. 43: 436-447 and KREMSDORF etal, 1983, J. Virol. 48: 340-351). These HPV have been divided into threegroups as a consequence of the absence of cross-hybridization or veryweak cross-hybridization between the genomes of the different groups.The first group includes the HPV3a and 10 which are associated withplane warts observed in some patients suffering from EV and in thepopulation generally: DNA sequences similar to those of HPV3a have beenfound in a cancer of a patient suffering from EV. The second groupcomprises HPV5, 8 and 12, the genomes of HPV5 and 8 having been detectedin cancers of patients suffering from EV. The third group is constitutedat the moment by a single virus, HPV9. With the exception of a recipientof a renal allograft presenting immunosuppression and who was shown tobe infected with HPV5, the viruses of the last two groups had only beendetected in patients suffering from EV, most of them being infected withseveral viruses. It is to be noted that, of the 14 types of HPVpresently mentioned in the literature (literature references 1-5, 8, 9,13, 14, 16, 18-20 listed at the end hereof), four were shown to bespecifically associated with EV, which is a rare disease.

The experiments which haveled to the invention and which have made itpossible to isolate a large number of new types and subtypes ofpapillomaviruses offer the promise of more refined, in vitro diagnosticprocedures. More particularly, the invention provides advancedprocedures for the identification of papillomaviruses isolated. forexample, from lesions or biopsy sections and makes it possible to carryout more precise diagnoses which are likely to result in improvedprognoses relative to the possible course the lesions in question maytake.

As a general rule, it will be noticed that the papillomaviruses.although differing very much from each other, have molecular sizes ofthe order to 7,000-8,000 base pairs. In addition, their genomes dononetheless exhibit certain degrees of homology. In what follows,reference will be made to assessments of the percentages of homologybetween types and subtypes of papillomaviruses, these percentages ofhomology being derived from hybridization assays carried out undernon-stringent conditions and, also, under stringent conditions.

Several types of papillomaviruses are distinguished by the percentagesof homology measured under stringent conditions. It is said that thepapillomaviruses which exhibit homologies of less than 50% under theseconditions belong to different types. It will be noted in this respectthat the percentages of homology between viruses of different types mayeven fall to zero under the said stringent conditions. Viruses for whichpercentages of homology higher than 50% are observed under thesestringent conditions are considered to belong to the same type and maygive rise to different subtypes within this same type.

The hybridization assays under non-stringent conditions involve placingin mutual contact DNAs derived from two isolates of virus under theconditions described by BEILDIAN, C. A. et al. 1980, J. Virol., 36,395-407, and CROISSANT et al., 1982, C. R. Acad. Sci. Paris, 294,581-586 (heteroduplex molecules).

Performance of hybridization assays under stringent conditions involvesplacing in mutual contact DNAs derived from two isolates of virus underthe conditions described by KREMSDORF, D. et al. ((1982), J. Virol. 43;436-447 and 1983, J. Virol., 48, 340-351) and DAVIS, R. W. et al., 1971,Methods Enzymol., 21, 413-418 (heteroduplex molecules)

Briefly, it will be noted that the papillomaviruses belonging to thesame type possess hybridizable sequences having nucleotide sequencesmore or less identical over 80% to 100% of their respective, totallengths, whereas such homologous sequences can be reduced to 60% or evenless in papillomaviruses of different types. The degree of identity oranalogy of the sequences of papillomaviruses of different types whichhybridize with each other under non-stringent conditions is obviouslylower than in the case in which the papillomaviruses belong to the sametype.

The study which the inventors have carried out has shown that both thedegree of genetic heterogeneity between papillomaviruses of differenttypes is greater than was hitherto considered to be the case and, at thesame time, that the different types are often found associated withforms or variants of infections presenting a certain degree ofspecificity.

Consequently, the invention relates not only to DNAs prepared from thevarious new papillomaviruses which have been isolated and the probeswhich may be constituted by all or part of these DNAs, but also mixturesor "cocktails" of types of papillomaviruses which may be put to use forthe more effective diagnosis of various types of infection, and even forthe diagnosis of the level of risk which accompanies the discovery in apatient of specific types of papillomaviruses. The number of probes forpapillomaviruses described in the present application to which may beadded, if necessary, those composed of genomic DNAs of papillomaviruseswhich bad already been isolated and their combination in definedmixtures thus making it possible to perform more refined diagnoses, inparticular, by enabling a clearer distinction to be made between thevarious types of infection and the various types of papillomavirus towhich they may be ascribed, or the infections which are liable todevelop as a result of the influence of such papillomaviruses,and,within a given class of specific infections, by providing a betterdiagnosis of the degree of risk of these infections being transformedinto more serious diseases. For example, the aim of the invention is toprovide agents which make it possible, in cases of infectionsmanifesting themselves as cases of epidermodysplasia verruciformis, toassess more precisely the degree of risk that the latter will developinto epidermal cancers.

As a general rule and with the aim of simplifying the subsequentdescription, the entire genomes of papillomaviruses will be designatedby the abbreviation "DNA-HPV".

With the same aim of simplifying the text in mind, reference is made inthe subsequent discussion to diagrams in which the figures consist ofrestriction maps of the DNA-HPVs, which include, moreover. DNA-HPVs ofpapillomaviruses already known.

The restriction maps give the positions of the cleavage site of variousrestriction endonucleases. The origin of the maps is usually constitutedby a unique cleavage site. The distances from the origin are expressedin percentages of the length of the genome, One map unit represents 1%of the length of the genome.

In the first place, the invention relates more particularly to each ofthe DNA-HPVs chosen from among the totality of DNAs which possessmolecular sizes varying between 7,000 and 8,000 base pairs and which, inrespect of the DNA-HPVs obtained from papillomaviruses, arecharacterised by restriction maps which appear in the diagrams and whichhave been given the designations HPV-2d, HPV-10b, HPV-14a, HPV-14b,HPV-15, HPV-17a, HPV-17b. HPV-19, HPV-20, HPV-21, HPV-22, HPV-23,HPV-24, HPV-28, HPV-29, HPV-31 and HPV-32, HPV-IP2 and HPV-IP4.

It is obvious that the invention also extends its coverage to DNA-HPVswhich can be considered as belonging to the same types as those whichhave just been mentioned.

The restriction maps corresponding to the DNA-HPVs of recentlycharacterized viruses are indicated by a closed black circle. Theinvention also relates to the aforementioned DNA-HPV fragments or tofragments capable of hybridizing with these latter, in particular, understringent conditions. Similarly, the invention relates to recombinantDNAs containing all or part of each of the DNA-HPVs cited above, andmore especially to DNA recombinants containing fragments correspondingto the genes E1, E6-E7, L1 and L2 and also fragments containingsequences corresponding to the intergenic regions of the said DNA-HPVs.The invention finally relates to the probes which can be constitutedfrom these respective DNA-HPVs or from corresponding fragments, and thein vitro diagnostic procedures in which these probes are used.

The preparations of viral DNA were extracted selectively (LUTZNER, M. A.et al., 1983, Lancet ii: 422-424) from material obtained by grattage ofbenign lesions in six European patients suffering from EV and in twoSouth American patients suffering from EV. The DNAs of HPVs werepurified by equilibrium centrifugation in cesium chloride gradientsand/or sedimentation in sucrose gradients in the presence ofethidiumbromide, in accordance with experimental procedures previouslydescribed (the articles of KRE2MSDORF, et al. cited above and ORTH, G,et al. 1980, Cold Spring Harbor Conf. Cell Proliferation 7:259-282). Thepreparations of DNA were treated with restriction endonucleases and thedigestion products were separated by electrophoresis from agarose gels(the articles by KREMSDORF et al. already cited). In addition to theHPV5, 8 and 12 (the articles by KREMSDORF et al. already cited) and HPV2(HEILMAN, C. A. et al., 1980, J. Virol. 36:395-407 and ORTH, G. et al.,1980, Cold Spring Harbor Conf. Cell Proliferation 7: 259-282) isolatedfrom the verrucae of one of the patients, eleven strains have beenidentified which provide important models of cleavage of DNA byrestriction enzymes, different from those which have been previouslycharacterized. The new types of HPV have been assigned a number and thesubtypes of a given type have been assigned the same number followed bya letter, denoting the chronological order of their identification(COGGIN, J. R. et al., Cancer Res. 39:545-546). The genomes of the 11new HPVs have been cloned in Escherichia coli K12, strain C600 (articleby KREMSDORF, D et al., (1983) already cited). The DNAs were inserted inthe form of molecules of unit length with the exception of two fragmentsof the DNA of HPV24 produced by the endonuclease BamHI. They wereinserted either into the plasmid pBR322 (SUTCLIFFE, J. G., 1978, NucleicAcids Res. 5:2721-2728) by making use of the unique cleavage sites ofAvaI, BamHI and HindIII, or into the recombinant plasmid which hasintegrated the HindIII B fragment of the DNA of HPV5 (article byKREMSDORF, D. et al., 1982, already cited) which contains a unique SacIsite. More particularly, the HPV17b and 22 were inserted in the form ofDNA molecules of unit lengths after being cleaved with an enzyme (SacI)which cleaves the HPV-DNA and the recombinant plasmid pBR322 containingthe HindIII B fragment of the DNA or HPV5 only once. The DNA of HPV14awas inserted into the plasmid pBR322 in the form of a DNA molecule ofunit length after partial digestion of the viral DNA preparation withHindIII, an enzyme which produces two fragments of 96.1% and 3.9% of thelength of the genome. The BamHI A and B fragment of HPV24 (which havesizes corresponding to the numbers 83.1% and 16.9% of the length of thegenome, respectively) were inserted separately into the plasmid pBR322.

The clones isolated and the sources of the corresponding HPV arepresented in Table I which follows:

                                      TABLE I    __________________________________________________________________________    ORIGIN OF THE CLONED HPV-DNAs.                                  Other types of              Type of HPV-DNA     HPV found in the    Patient.sup.a        Nationality              Source.sup.b                      cloned                          Cloning enzyme.sup.c                                  patients    __________________________________________________________________________    1   Polish              warts; knees                      14a Hind III                                  5                      15  Bam HI    2   French              warts; hands                      14b Bam HI    3   Colombian              maculae; trunk                      17a Bam HI  5    4   Italian              maculae; chest                      17b Sao I   5                      22  Sao I    5   Dutch maculae; back                      19  Bam HI  5, 8, 17a              maculae; chest                      24  Bam HI    6   Colombian              warts; hands                      20  Ava I   5, 8, 24    7   Polish              warts; knees                      21  Bam HI  2, 12, 17a, 20    8   Polish              maculae; forearm                      23  Bam HI  5, 8, 20    __________________________________________________________________________

In order to identify the recombinant plasmids the electrophoreticmobilities of the digestion products of the recombinant DNAs and thenon-cloned HPV-DNAs were compared after treatment with a mixture of tworestriction endonucleases containing the endonuclease utilized to insertthe viral sequences into the plasmid. The number and the molecular sizeof the isolated fragments indicated that the entire viral genomes hadbeen integrated in each case. Size heterogeneity of the DNAs wasobserved when the DNAs of the HPVs non-cloned or excised from theplasmid. sequences, were analysed by electrophoresis on agarose gel(data not presented). The DNAs of HPV14b, 19, 20 and 21 have sizessimilar to those of HPV3a, 5, 8 and 12 (about 7,700 ntucleotide pairs)(articles by KREMSDORF, D. already cited), whereas the DNAs of HPV15,17a, 17b, 22 and 23 are somewhat less like that of HPV9 (about 7,200nucleotide pairs) (articles by KREMSDORF, D., 1982 and ORTH, G., 1980,already cited).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2, 4, 4, 5, 6, 7, 8, 9 and 10 represent restriction maps of HPVviral genomes.

FIG. 11 shows steps for production of L2 protein.

The sensitivity of the cloned viral genomes to 14 restrictionendonucleases has been analysed and the corresponding restriction mapshave been established (FIGS. 1 to 10). The restriction maps of some ofthe HPV-DNAs-are reproduced in some of the figures for reasons whichwill be explained later. Between 22 and 33 cleavage sites have beenlocalized by means of the methods previously described (9). No obviousanalogy was detected between these maps, with the exception of those ofHPV14a and 14b, on the one hand (FIGS. 4a and 4b), and those of HPV17aand 17b, on the other (FIG. 5). Of the 21 and 31 sites localized on theDNAs of the HPV14a and 14b, respectively, 15 were found to be common toboth when one of the two BamHI cleavage sites of the DNA of HPV14a wasaligned with the unique BamHI cleavage site of the DNA of HPV14b. In asimilar manner, 21 of the 29 cleavage sites situated on the DNA ofHPV17a were also found to correspond to sites on the DNA of HPV17b (with26 sites) when the unique BamHI cleavage sites were aligned.

No obvious analogy was detected between these maps and those previouslyestablished for the HPV associated with EV (HPV3a , 5, 8, 9, 10 and 12)(8, 9, 16, 18, 20), with cutaneous warts (HPV1, 2, 4 and 7), and withlesions to mucous membranes (HPV6b, 11a, 13 and 16) (1, 33, 19), withthe exception of the map of HPV14a which is closely related to the mapof an HPV isolated from a Japanese patient suffering from EV (24). Thislatter isolate differs from HPV14a by two additional cleavage sites, onefor BamHI and the other for HindIII, whereas the locations of the AvaI,BamHI, BghI, EcoRI, HindII and HindIII sites are similar for the twoviruses. Cross-hybridization experiments have confirmed that these twoviruses are very closely related.

It will also be noticed that some sites (those indicated by the arrows)have not been localized. Cleavage sites which differ in their locationby less than 2% of the length of the genome are considered to beconserved (). The enzymes which did not give rise to any cleavage were:PvuI, SalI and SmaI for the DNAs of HPV14a and 23; PvuI, SacI, SalI andSmaI for the DNA of HPV14b; BglI, PvuI, SalI and SmaI for the DNAs ofHPV15, 17a and 17b; BglI, SacI, SalI and SmaI for the DNA of HPV19;EcoRI, PvuI, SacI and SmaI for the DNA of HPV20; SacI and SmaI for theDNA of HPV21; BamHI, BglI, PvuI, PvuII and SalI for the DNA of HPV22;BglI, EcoRI, PvuI, SacI, SalI and SmaI for the DNA of HPV24.

The existence of sequence homology between DNAs of recentlycharacterized HPV as well as between these latter and the DNAs of theHPV of EV previously characterized (HPV3a , 5, 8, 9, 10 and 12), of theHPV associated with dermaiwarts (HPV1, 2, 4 and 7), and of HPVassociated with lesions to mucous membranes (HPV6b, 11a, 13 and 16) hasbeen studied. Hybridization experiments by fixation on filter paper andDNA-DNA hybridization in liquid phase at saturation followed bydigestion with the nuclease S1 were carried out under the stringentconditions previously described (8, 9). In particular, the HPV-DNAs werelabelled by "nick-translation" and fractionated by sedimentation inalkaline sucrose gradients (as numbers 5% to 20%)as previously described(13). HPV-DNAs (4,000 cpm) were incubated in 0.48 M NaCl--1 mM EDTA (pH6.8) at 68° C., in the presence of either DNA of calf thymus (20 μg) ornon-labelled HPV-DNA (0.20 μg) as previously described (8, 9). Thespecific activities of the HPV-DNA probes varied between 5.3×10⁷ and2×10⁸ cpm/μg. The percentage of hybridization was determined bymeasuring the fractions resistant to the nuclease S1. The numbersrepresent the values corrected for the spontaneous auto-renaturation ofthe probes (4% to 15%) and normalizedto 100% for homologoushybridization (75% to 95%). The abbreviation ND means not determined.The relative extent of cross-hybridizations between the HPV-DNAs underthe conditions mentioned above are expressed as a percentage ofhybridization between a labelled HPV-DNA and an unlabelled HPV-DNA.

                                      TABLE 2    __________________________________________________________________________    DEGREE OF CROSS-HYBRIDIZATION BETWEEN THE HPV-DNAs DETERMINED IN    SOLUTION    Unlabelled          % hybridization with .sup.32 P-labelled HPV-DNA    HPV-DNA          3a 5  14a                   14b                      19 20 21 22 23 9  15 17a                                              17b                                                 24    __________________________________________________________________________     1a   0.1             0.3                0.2                   0.3                      0  0.8                            2.9                               0  0  1.0                                        0.4                                           0.2                                              0  0    11a   1.6             1.0                1.3                   0  0.1                         0.1                            0.7                               3.7                                  0  0.1                                        0.1                                           0.6                                              3.3                                                 0     3a   100             1.8                1.0                   0  1.5                         0.1                            1.5                               0  1.9                                     0.1                                        1.7                                           1.2                                              1.8                                                 3.0    10    32.3             ND ND 0.1                      0  0.1                            1.9                               3.0                                  ND 0  1.6                                           0.1                                              2.0                                                 ND     5    0.2             100                12.1                   12.4                      5.8                         9.3                            9.4                               10.1                                  5.8                                     4.3                                        0.7                                           3.6                                              0  2.4     8    1.1             15.7                9.9                   13.4                      8.5                         5.6                            5.0                               7.1                                  5.8                                     3.5                                        1.5                                           3.2                                              3.8                                                 0.1    12    0.1             19.3                9.2                   12.5                      5.3                         8.6                            9.3                               11.7                                  4.0                                     3.6                                        1.2                                           0.1                                              1.9                                                 0    14a   0.2             13.2                100                   88.8                      14.6                         12.4                            32.9                               10.1                                  24.6                                     3.0                                        2.2                                           2.4                                              4.1                                                 ND    14b   ND 10.5                94.1                   100                      9.3                         28.4                            35.4                               9.5                                  28.2                                     0  0  0  0  0    19    ND 7.2                21.4                   20.6                      100                         7.6                            8.8                               15.5                                  27.7                                     0  0  0  2.2                                                 1.0    20    ND 9.9                28.8                   37.9                      6.2                         100                            25.4                               13.7                                  14.1                                     0  0  2.1                                              0  3.6    21    ND 10.5                38.7                   40.5                      6.4                         37.5                            100                               9.8                                  18.6                                     0.1                                        0  2.5                                              0.3                                                 0    22    ND 7.2                7.4                   ND 17.3                         7.2                            10.0                               100                                  17.9                                     0  0  0.1                                              0.1                                                 0    23    ND ND ND ND ND ND ND 21.2                                  100                                     0  0.5                                           0  0  0.1     9    0.4             3.1                0.5                   1.2                      0  2.0                            1.0                               0  0  100                                        5.5                                           6.3                                              5.4                                                 0    15    0.4             3.3                2.1                   3.3                      0  0.1                            0.8                               0  0  7.8                                        100                                           22.5                                              21.6                                                 0    17a   0.7             1.2                1.4                   2.8                      0  0.1                            0.1                               0.8                                  1.4                                     7.6                                        19.5                                           100                                              92.7                                                 0    17b   ND ND ND 1.4                      0  0.3                            3.4                               ND ND ND ND 86.3                                              100                                                 ND    24    ND ND 0.1                   2.6                      ND ND ND 0.8                                  0  0.2                                        0  1.1                                              1.1                                                 100    __________________________________________________________________________

It will be noted that there is an absence or virtual absence ofcross-hybridization between the genomes of HPV1, 2, 4, 6b, 7 and 11a andthe DNAs of the newly cloned HPV of EV labelled with ³² P or betweenDNAs of non-labelled HPV of EV and specific probes for HPV13, 16 and 18.Similarly, no or virtually no cross-hybridization was detected betweenthe DNAs of the HPV14a, 14b, 15, 17a, 17b, 19, 20, 21, 22, 23 and 24 andthe DNAs of the HPV1a and 11a by reassociation at saturation (Table 2).The newly cloned HFV-DNAs showed no or virtually no cross-hybridizationor a cross-hybrization of less than 50% among themselves and with thegenomes of the other HPV associated with EV (HPV3a, 5, 8, 9, 10 and 12),with the exception of the HPV14a and 14b, on the one hand, and theHPV17a and 17b, on the other, which showed considerablecross-hybridization. These observations justify the classification ofthe new viruses into nine new types (HPV14, 15, 17, 19, 20, 21, 22, 23and 24) plus two subtypes of the types 14 (HPV14a and b) and 17 (HPV17aand b).

Similarly, the various HPVs were classified in groups on the basis oftheir sequence homology (or absence of sequence homology) understringent conditions of molecular hybridization. These groups,designated by the letters A to H, are listed in Table III which follows.This table indicates the diseases which were diagnosed in the carriersof these HPVs (in isolation or in combination) and the oncogenicpotential which is ascribed to each of them.

The DNAs of the HPV5, 8, 12, 14, 19, 20, 21, 22 and 23 exhibit degreesof cross-hybridization (group homologies) varying from 5% to 38%, andexhibit a noteworthy degree of cross-hybridization (4% to 13%) only withthe DNAs of the HPV5, 8 and 12. Thus, these viruses form part of a groupof HPV of EV previously defined (9).

Similarly, the DNAs of the HPV9, 15 and 17, which display amongthemselves a cross-hybridization of about 20% and a cross-hybridizationof about 6% with the DNA of HPV9, also belong to a group of HPV of EValready described (9). The HPVs types 13 and 31 may be considered asbelonging to the same group. Finally, the HPVs types 1, 2, 4, 24 and 32,which exhibit almost no homology with the genomes of the other HPV areconsidered to represent the first numbers of other groups, distinct fromeach other and from the groups already known.

The invention also relates more especially to fragments of DNA, derivedfrom the above-mentioned HPV-DNAs, and more particularly to

                                      TABLE III    __________________________________________________________________________    CLASSIFICATION OF THE HPV WHICH ARE THE SUBJECT OF THE PATENT    APPLICATION    ACCORDING TO THE DEGREE OF HOMOLOGY OF THEIR NUCLEOTIDE SEQUENCE    DETERMINED    BY MOLECULAR HYBRIDIZATION UNDER STRINGENT CONDITIONS               Homologies        Types.sup.2)               within the                      Mixture    Group.sup.1)        of HPV group Associated diseases                                   Oncogenic potential                                               of probes    __________________________________________________________________________    A   1            myrmecias     very weak   1    B   2            verrucae      weak        1    C   3, 10, 28*, 29*               14 å 38%                     plane warts   moderate    2                     intermediate warts                                   a related virus,                                               2                     actinic keratoses                                   assocaited with intra-                     Bowen's disease                                   epithelial neoplasias and                                   and cutaneous cancers,                                   characterization pending    D   4            verrucae      very weak   1    E   5.0, 12, 14*               4 a 38%                     epidermodysplasia verruci.                                   HPV5, 8 and 14 associated                                               3, 4, 7        19*, 20*, 21*,                     actinic keratoses                                   with EV carcinomas; a        22*, 23*     Bowen's disease                                   related virus, associated                     cutaneous carcinomas                                   with intra-epithelial                                   neoplasias and cutaneous                                   cancers, characterization                                   pending    F   9, 15*, 17*               6 a 23%                     Epidermodysplasia verruciformis                                               5    G   24*          Epidermodysplasia verruciformis                                               6    H   13, 31*      Oral epithelial hyperplasia.                                               8                     Oral leucoplasias    I   32*          Bowen's disease                                   cutaneous intra-epithelial                                               7, 9                                   neoplasias    __________________________________________________________________________     .sup.1) The genomes of the HPV types belonging to different groupos     usually do not show detectable sequence homology under stringent     conditions of molecular hybridiation. The genomes of the HPV types     belonging to the same group show a sequence homology of less than 50%.     .sup.2) The new HPV types are indicated by an asterisk.

those corresponding to the genes E6-E7; E1; L2; L1 and to theirintergenic regions. The relative position and length of these variousfragments with respect to the sites taken as origins (FIGS. 1 to 9) areindicated in Table IV which follows:

                  TABLE IV    ______________________________________    PUTATIVE LOCALIZATION OF THE PRINCIPAL GENES AND    OF THE INTERGENIC REGION ON THE PHYSICAL MAPS OF    THE HPV GENOMES    Co-ordinates of the 5' and 3' ends of the fragments    corresponding to    HPV  genes                   the intergenic    type E6-E7    E1       L2     L1     region    ______________________________________     1     44-34.5                  35-11      95-75.5                                  76.5-56                                           56-44.5     3   18.5-9    9.5-85.5                           69.5-50                                    51-30.5                                         30.5-19     5   6.5-97   97.5-73.5                           57.5-38                                    39-18.5                                         18.5-7     8     63-53.5                  54-30      14-94.5                                  95.5-75                                           75-63.5     9     42-32.5                  33-9       93-73.5                                  74.5-54                                           54-42.5    10a    49-39.5                  40-16      0-80.5                                  81.5-61                                           61-49.5    10b    93-83.5                  84-60      44-24.5                                  25.5-5   5-93.5    12   23.5-14  14.5-90.5                           74.5-55                                    56-35.5                                         35.5-24    14   8.5-99   99.5-75.5                           59.5-40                                    41-20.5                                         20.5-9    15   39.5-30  30.5-6.5 90.5-71                                    72-51.5                                         51.5-40    17     46-36.5                  37-13      97-77.5                                  78.5-58                                           58-46.5    24   24.5-15  15.5-91.5                           75.5-56                                    57-36.5                                         36.5-25    28   47.5-38  38.5-14.5                           98.5-79                                    80-59.5                                         59.5-48    29   89.5-80  80.5-56.5                           40.5-21                                   22-1.5                                         1.5-90    31     89-78.5                    80-53.5                           33.5-15.5                                  17.5-96.5                                         96.5-92.5    ______________________________________

The localization of the genes on the genome of HPV1 was deduced from thenucleotide sequence of this genome (Patent O. Danos, M. Katinka and M.Yaniv). The physical maps of the genomes of the HPV3, 5, 8, 9, 10a, 12,14, 15, 17 and 24 were aligned with the physical map and the genetic mapof HPV1, and that of HPV31 with the physical map and the genetic map ofHPV6b (E. Schwarz et al, EMBO J., 1983, 2, 2341-2348), after analysis inthe electron microscope of heteroduplex molecules formed under stringent(Tm -29° C.) or less stringent (Tm -40° C.) conditions of hybridization.The physical maps of HPV10b, 28 and 29 were aligned with the physicalmaps of HPV3a and 10a after the conserved sites for restriction enzymeshad been juxtaposed.

The values of the co-ordinates reported in Table IV show the position onthe physical maps presented in the FIGS. 1-9 of the 5' and 3' ends ofthe segments of the homologous genomes of the genes E6 and E7, E1, L2and L1 and of the intergenic region with respect to the genome of HPV1aor, in the. case of HPV31, with respect to the genome of HPV6b.

The intergenic region (comprising the elements concerned withregulation) and the adjacent genes E6 and E7 (which probably correspondto the principle transformation genes expressed in tumours do not showany sequence homology between genomes of HPV types belonging todifferent groups which is detectable by analysis in the electronmicroscope of heteroduplex molecules formed under non-stringentconditions of hybridization, or with genomes of most of the HPV typesbelonging to the same group when heteroduplex molecules formed understringent conditions are similarly analysed. The E1 gene (implicatedmainly in the replication of the viral DNA) and the L1 gene (coding forthe major protein of the viral capsid which bears the principalantigenic determinants of the virions) do present sequence homologiesbetween genomes of HPV types belonging to different groups which aredetectable by analysis of heteroduplex molecules formed undernon-stringent conditions of hybridization, and between genomes of HPVbelonging to the same group when heteroduplex molecules formed understringent conditions of hybridization are similarly analysed.

Probes prepared from recombinant plasmids containing the E1 and L1regions can theoretically be used to detect the largest number of HPVtypes by molecular hybridization experiments carried out under eitherstringent or non-stringent conditions. Probes prepared from recombinantplasmids comprising the intergenic region or the E6 and E7 genes may beused to specifically detect one type of HPV or closely related HPVtypes.

The L2 region (which codes for a minor component of the viral capsid)displays a variable degree of conservation of nucleotide sequences amongthe different HPV types.

In the discussion which follows more precise descriptions of theconditions under which the viruses HPV-IP2 and HPV-IP4 were isolatedwill be given, and these will be followed by a description of theconditions under which the HPV-DNAs were isolated from these viruses.

Molecular cloning and characterization of a novel type of HPV associatedwith neoplasias and genital cancers (HPV IP2).

The presence of a new type of HPV was demonstrated in the DNA extractedfrom a cancer of the uterine cervix by hybridization with a radioactiveprobe specific for HPV type 16 under non-stringent conditions. Nocross-hybridization was detectable when the hybridization was carriedout under stringent conditions of hybridization. A study of thesensitivity of the DNA of this HPV to several restriction enzymes showedthat the enzyme BglII cleaves the viral DNA at a unique site. Afterdigestion of the DNA extracted from the tumour with endonuclease BgIII,the fraction containing the DNA molecule of 8 kb (the size of apapillomavirus genome) was purified by centrifugation in a sucrosegradient. The molecules of 8 kb were inserted at the BglII site into avector constituted by the plasmid PL15.5 (which contains a uniquecleavage site for BglII and BamHI) which was inserted through its BamHIsite into the DNA of the bacteriophage lambda L47.1. After encapsidationof the recombinant DNA and infection of host bacteria (Escherichia coli,strain LA101), the lysis plaques corresponding to recombinant phageswere detected by Southern hybridization of the infected bacterialcultures using a radioactive DNA of HPV16 under non-stringentconditions. Several recombinant bacteriophages containing the totalityof the viral sequences were isolated: cleavage of the phage DNA by therestriction enzyme BglII leads to an 8 kb fragment which hybridizes withthe HPV16 probe under non-stringent conditions; cleavage of the DNA ofthe recombinant phages and the DNA of the original tumour by a mixtureof the enzymes BglII and PstI gives rise to the same 5 fragments, thesum of the molecular weights of which is equal to the molecular weightof the papillomavirus genome. The DNA of the novel HPV was excised fromthe DNA of the recombinant bacteriophages, purified by electroelution,and recloned in the plasmid PL15.5. A restriction map of the viral DNAwas constructed on the basis of the sensitivity of this DNA to 18restriction endonucleases the action of which led directly to theidentification of 21 cleavage sites.(FIG. 9). The map thus establishedwas different from the map of the genomes of the HPV identified up tonow. The sequence homology between the DNA of the novel HPV and the DNAof the HPV so far identified was analysed by Southern hybridizationexperiments carried out under stringent conditions. The homologydetected was always less than 5%, the greatest homology being detectedwith the genome of HPV16. The new virus, which was characterized from acancer of the uterine cervix, thus constitutes a new type of HPV,provisionally named HPV-IP2.

The electron microscopic analysis of heteroduplex molecules formed underdifferent conditions between the DNA of HPV-IP2 and the DNA of HPV1 hasmade it possible to align the physical maps of these two genomes and todefine the theoretical position of the different genes carried by theDNA of the HPV-IP2.

PUTATIVE LOCALIZATION OF THE PRINCIPAL GENES AND THE INTRAGENIC REGIONOF HPV-IP2 ON THE MAP OF THIS GENOME

    ______________________________________                    Co-ordinates of the ends                    5'   3'    ______________________________________    E6-E7             62     71.5    E1                71     95    E2                95.5   11.5    L2                11     30.5    L1                31.5   52    Intergenic region 52     63.5    ______________________________________

The use of radioactive probes prepared from the purified DNA of HPV-IP2has enabled the pathogenicity of these viruses to be determined. Thepresence of the DNA of HPV-IP2 was demonstrated in one case of bowenoidpapules of the external genital organs out of the 14 cases studied, intwo invasive cancers of the uterine cervix out of 51 cases studied andin one case of intra-epithelial neoplasia of the uterine cervix out of28 cases studied. Thus, HPV-IP2 represents a HPV type showing genitaltropism and displaying oncogenic potential, the frequency of which issome what less than that of HPV18, and markedly less than that of HPV16.It is necessary to incorporate it in all mixtures of HPV-DNA intendedfor the preparation of molecular probes with a view to the diagnosis orscreening of HPV types consituting a risk of developing into genitalneoplasias and, in particular, cancers of the uterine cervix.

Molecular Cloning and Characterization of a Novel Type of HPV Associatedwith Precancerous Lesions of the Skin (HPV-IP4).

A novel type of HPV was demonstrated in the DNA extracted from a biopsyof actinic keratosis, a precancerous skin lesion, by molecularhybridization with a mixture of radioactive probes specific for the HPVtypes 5, 8 and 14 under stringent conditions. No cross-hybridization wasdetected when the hybridization was carried out with probes specific forthe types 1, 2, 3, 7, 10, 13, 16, 18, 28, IP1 (previously known asHPV31), IP2 and IP3 (previously known as HPV32).

A study of the sensitivity of the DNA of this HPV to several restrictionenzymes has shown that the enzyme EcoRI cleaves the viral DNA once.After digestion of the DNA extracted from the biopsy from theendonuclease EcoRI, the fraction containing the DNA molecules of 8 kb(corresponding to the size of the papillomavirus genome) was purified bycentrifugation in a sucrose gradient. The 8 kb molecules were insertedat the EcoRI site into the DNA of the bacteriophage λgt wes λB. Afterencapsidation of the recombinant DNA and infection of host bacteria(Escherichia coli, strain LA101), the lysis plaques corresponding torecombinant phages were detected by Southern hybridization of theinfected bacterial cultures using a mixture of the radioactive DNAs ofHPV5, 8 and 14 under non-stringent conditions. Several recombinantbacteriophages containing the totality of the viral sequences wereisolated: cleavage of the phage DNA by the restriction enzyme EcoRIgives rise to an 8 kb fragment which hybridizes with the probe specificfor HPV5, 8 and 14 under non-stringent conditions; cleavage of the DNAof the recombinant phages and the DNA from the original lesion by amixture of the enzymes EcoRI and PstI gives rise to the same sixfragments, the sum of the molecular weights of which is equal to themolecular weight of the papillomavirus genome. The DNA of the novel HPVwas excised from the DNA of a recombinant bacteriophage, purified byelectroelution, and recloned in the plasmide pSP65. A restriction map ofthe viral DNA was constructed on the basis of the sensitivity of thisDNA to 15 restriction endonucleases, the action of which led directly tothe identification of 23 cleavage sites (FIG. 10). The map thusestablished is different from the map of the genomes of the HPVsidentified up to now. The sequence homology between the DNA of the novelHPV and the DNA of the HPVs so far identified was analysed by Southernhybridization experiments carried out under stringent conditions. Ahomology of less than 50% was detected between the DNA of the novel HPVand the DNA of certain types of HPV previously identified in lesions ofepidermodysplasia verruciformis (HPV5, 8, 12, 14, 19, 20, 21 and 25),but no homology was detected with the other types of HPV. The new viruscharacterized from an actinic keratosis isolate thus constitutes a newtype of HPV, provisonally referred to as HPV-IP4.

The use of a radioactive probe prepared from the purified DNA of HPV-IP4has led to the demonstration of the presence of HPV-IP4 in 42% of 17patients studied who were suffering from epidermodysplasia verruciformisand in biopsies of actinic keratosis analysed. In view of its highfrequency in patients suffering from epidermodysplasia verruciformis, adisease characterized by the frequent development of skin cancers, andas a consequence its association with a fraction of actinic keratosislesions which are considered to be precursors of squamous cell carcinomaof the skin, HPV-IP4 constitutes a type of HPV showing dermal tropismand displaying oncogenic potential. It is necessary to incorporate it inall mixtures of HPV-DNA intended for the preparation of molecular probeswith a view to the diagnosis or screening of types of HPV constitutinrisk of developing into cancerous or precancerous lesions of the skin.

The invention also relates more especially to mixtures or cocktails ofdifferent HPV-DNAs (or probes containing such HPV-DNAs or fragments ofthe latter), combinations which can be used to carry out comprehensivediagnoses of different forms of papillomavirus infections, the ultimateaim of which is to provide a prognosis of the possible course theinfection will take. Preferred mixtures in accordance with the infectionare presented in Table V which follows.

This table also indicates the natures of the diseases most likely to bediagnosed by the use of the mixtures appearing at the left-hand side ofthe table. It will be noted that the groups in which the restrictionmaps are presented in the accompanying FIGS. 1-9 correspond to those ofthe viral types indicated in the column "Constitution" of Table V. Thisis also the reason which has led to some of the probes being reproducedseveral times in different figures of the appendices.

Each of these mixtures may also be defined as comprising at least one ofthe novel probes according to the invention. In other words, themixtures for diagnosis according to the invention may be defined ascontaining:

1) at least the DNA of FPV2d,

2) at least one of the DNAs of HPV10b, 28 and 29,

3) at least one of the DNAs of HPV17, 24,

4) at least one of the DNAs of HPV14, 15, 17, 19, 20, 21, 22 and 23,

5) at least one of the DNAs of HPV15 and 17,

6) the DNA of HPV24,

7) the DNA of HPV14, 32,

8) the DNA of HPV31,

9) the DNA of HPV32,

it being understood that the DNAs of the nine groups are chosen in amanner so as to be different from each other under all circumstances.

Given the great variety of HPVs which are likely to be isolated fromdifferent forms of warts or other mucosal or cutaneous lesions, it ispreferable to use mixtures containing more than one or two HPV-DNAs forthe diagnosis of each type of disease mentioned in the table as aconsequence of its being recognized that other HPV-DNAs may be involvedin the development of the same type of disease. The diagnosis

                                      TABLE V    __________________________________________________________________________    CHARACTERISTICS OF MIXTURES OF HPV-DNA UTILIZABLE FOR THE VIROLOGICAL    DIAGNOSIS OF    OF PAPILLOMAVIRUS INFECTIONS    Designation    of mixtures          Consitution.sup.1)  Disease to be diagnosed    __________________________________________________________________________    1     1, 2d*, 4           Cutaneous or mucosal warts                              (in particular, verrucae and plantar warts).                              Differential diagnosis of epidermodysplasia                              verruciformis    2     3, 10a, 10b*, 28*, 29*                              Mucosal, cutaneous, intermediary or plane                              warts.                              Intra-epithelial neoplasias and cutaneous                              cancers.                              Differential diagnosis of epidermodysplasia                              verruciformis.    3     5, 17a*, 24*        Epidermodysplasia verruciformis.                              Intra-epithelial    4     5, 8, 12, 14a*, 14b*, 19*, 20*, 21*, 22*, 23*                              Epidermodysplasia verruciformis.    5     9, 15*, 17a*, 17b*  Epidermodysplasia verruciformis.    6     24*                 Epidermodysplasia verruciformis.    7     5, 8, 14b*, 12*     Cutaneous cancers of epidermodysplasia                              verruciformis.                              Intra-epithelial neoplasias and cutaneous                              cancers.    8     13, 31*             Oral epitehlial hyperplasia; differential                              diagnosis                              of oral intraepithelial neoplasias.    9     32*                 Intraepithelial neoplasias and cutaneous                              cancers.    __________________________________________________________________________     .sup.1) The new HPV types introduced into the consitution of the mixtures     of probes are indicated by an asterisk.

of the nature of the disease and its possible course will be the moreefficacious, the higher the number of probes used. In addition.hybridization assays carried out with different mixtures of probesenable differential diagnoses to be made which, in turn, render morelikely the correctness of the diagnosis of the disease from which thepatient is suffering.

In Table V, only probes composed of HPV-DNAs isolated in the laboratoryof the inventors have been mentioned. It will be obvious, in view of thepreceding discussion, that the various mixtures can be advantageouslysupplemented with DNAs derived from HPVs isolated in other laboratories,should these viruses have been found on different occasions in patientssuffering from the same type of disease. For example, it can only be anadvantage for mixture 7 to be supplemented with all of the otherHPV-DNAs encountered in types of epidermodysplasia verruciformis whichrisk being transformed into intra-epithelial neoplasias and skincancers. It will be noticed in Table V that some of the mixtures arepresented as being characteristic for the diagnosis of identicaldiseases. Nonetheless, it is to be noted that different mixtures dodistinguish between infections with a low risk of carcinogenesis andinfections with a high risk of carcinogenesis. For example,hybridization occurring with a viral preparation derived from a patientsubjected to diagnosis with mixture 7 provides evidence of a higher riskof skin carcinogenesis than the case in which hybridization occurspreferably with mixture 3.

Similarly, the EV detected by mixture 5 provide evidence of a higherrisk of carcinogenesis than in EV detected by mixture 6. Mixture 4detects EV presenting an even higher risk than those detected by mixture5.

Other mixtures are cocktails of different HPV-DNAs (or probes containingthese HPV-DNAs or fragments of the latter) will be described below whichcan be used in combination to carry out comprehensive diagnoses ofdifferent forms of papillomavirus infections, ultimately with the aim ofproviding a prognosis of the possible course the infection may take.

Preferred mixtures in accordance with the invention are indicated inTable V mentioned above.

The above table also indicates the nature of the diseases more likely tobe diagnosed by the utilization of the mixtures presented at theleft-hand side of the table. It will be recalled that the restrictionmaps of the other HPV-DNAs identified in the preceding table arepresented in the FIGS. 1 to 9.

It is to be noted that HPV-IP2 may be considered as especiallyrepresentative of probes which may be used for the detection of the riskof the development of genital neoplasias and, in particular, of cancersof the uterine cervix.

Thus, the invention relates more especially to diagnosis "kits"comprising at least ten groups appearing in the groups numbered 1 to 10in the table under the heading "Designation of mixtures".

In the foregoing, particular consideration has been given to the use, asprobes, of cloned, whole HPV-DNAs. The latter may however be replaced bycloned fragments of these DNAs, in particular, by the genes E1 or L1 andby the genes E6-E7.

The basic principle of in vitro detection of HPV-DNA naturally dependson hybridizations carried out under stringent or less stringentconditions. For example, off may proceed as follows, provided, ofcourse, that it is understood that the diagnostic assays described arenot to be considered as limiting with respect to the conditions underwhich probes or mixtures of probes according to the invention are used.

The aim of the investigations in which probes prepared from mixtures ofcloned DNAs of HPVs are used is to demonstrate an HPV and to identifythe type of HPV in a biopsy, in cells obtained by grattage of lesions,or in biopsy sections fixed with Carnoy mixture (ethanol, chloroform,acetic acid: 6:3:1) and embedded in paraffin. The investigation requiresthat the DNA be first extracted from samples according to known methodsand involves the analysis of this DNA by molecular hybridizationexperiments, carried out under strict or less strict conditions usingradioactive probes (labelled with ³² P or ³⁵ S) prepared from mixturesof HPV-DNAs. Each investigation usually requires that several mixturesof probes be used.

Several methods of hybridization may be used. For example, the method ofdot blot hybridization may be employed. This method involves thedenaturation of the DNA and the depositing of aliquots of the DNA onmembranes (nitrocellulose or GENESCREENPLUS), the hybridization of eachmembrane under standard conditions with a mixture of probes and thedetection of the radioactive hybrids by exposure of the membrane to aradiographic film. The method of Southern hybridization may also beused. This method consists of the electrophoretic separation of the DNAfragments produced by treatment of the DNA with restriction enzymes inan agarose gel, the transfer of the fragments to membranes(nitrocellulose, GENESCREENPLUS) after alkaline denaturation and theirhybridization under standard conditions with different mixtures ofprobes. The formation of radioactive hybrids is detected after exposureof the membrane to radiographic film.

The radioactive probes are constituted either by the DNAs of HPVslabelled by the method of "nick-translation", or by RNAs prepared bytranscribing the viral DNAs inserted into a vector of the SP6 type, forexample. The use of radioactive probes offers the advantage of highsensitivity but that does not exclude the use of non-radioactive probes,biotinylated probes for example, capable of being recognized byantibodies either labelled themselves or recognized by antibodiesbearing a marker such as an enzyme, fluorescence label, etc . . .

The choice of probes depends on the nature of the samples. Thus, forexample, in the case of a patient suspected to be suffering from EV, themixtures 1, 2, 3, 4, 5, 6 and 7 will be used. The mixtures 1 and 2enable a differential diagnosis between EV and cutaneous warts to bemade. Probe 3, containing the most frequently detected member of each ofthe three groups of HPVs associated with the disease, and the probe 7,containing the DNAs of the types of HPV associated with EV cancers, willpermit the diagnosis of EV in the majority of cases and, in particularwill lead to the identification of the patients infected with the typesof HPV presenting risk of developing into cancers. The use of themixtures 4, 5 and 6 makes it possible to define more precisely the typeor types of HPV infecting this same patient.

Thus, the invention also relates to "kits" containing several probes ofthe type indicated above, in particular:

either representatives of each of the 19 types and subtypes of HPV-DNAsindicated above,

or mixtures of probes, preferably the various groups or mixtures ofprobes which had been defined above,

these "kits" being intended for in vitro diagnostic studies involvinghybridization between the viral preparations obtained from patients andthe various groups or mixtures.

It is obvious and also follows from the foregoing that the invention isin no way limited to those of its mode of application and realisationwhich have been given particular consideration; on the contrary, itincludes all possible variants; in particular, the reference in theclaims to a designation DNA-HPV, followed by a specific numbercorresponding to a DNA-HPV, the restriction map of which has beenprovided in the Figures, is to be understood as indicating that theseclaims cover all of the DNA-HPVs which have in common with thisparticular DNA-HPV properties allowing them to be classed as being ofthe same type, in accordance with the definition of the type given aboveand, with all the more reason, all of the DNA-HPVs which belong to thesame subtype.

With particular regard to the DNA derived from HPV32, it is to be noted,and this is made clear in the diagrams,that this DNA is not cleaved byAvaI, BalI, BamHI, ClaI, EcoRI, HindIII, NdeI, NruI, PvuI, PvuII, SacI,SalI, Smal, TthIII, XmaI.

It will be noted that the recombinant DNAs designated below weredeposited with the C.N.C.M. (Collection Nationale des Cultures deMicro-Organismes de l'INSTITUT PASTEUR de Paris) on the 30th of November1984 under the numbers which appear below:

pBR322/HPV2d . . . No. I-379

pBR3222/HPV10bA . . . No. I-380

pBR322/HPV10bB . . . No. I-381

pBR32/HPV14a . . . No. I-382

pBR322/HPV14b . . . No. I-383

pBR322/HPV15 . . . No. I-384

pBR322/HPV17a . . . No. I-385

pHPV5 HindIIIB/HPV17b . . . No. I-386

pBR322/HPV19 . . . No. I-387

pBR322/HPV20 . . . No. I-388

pBR322/HPV21 . . . No. I-389

pHV5 HindIIIB/HPV22 . . . No. I-390

pBR322/HPV23 . . . No. I-391

pBR322/HPV24a . . . No. I-392

pBR322/HPV24b . . . No. I-393

pBR322/HPV28 . . . No. I-394

pBR322/HPV29 . . . No. I-395

pBR322/HPV31 . . . No. I-396

pSP64/HPV32 . . . No. I-397

pLl55/IP2 . . . No. I-450

pSP65/IP4 . . . No. I-449

The invention also relates more particularly to the products expressedby the genes E6, E7 and especially L2 of the different paillomaviruseswhich have been mentioned in the discussion thus far.

These products of gene expression may themselves be used for thedetection of papillomaviruses or products expressed by them in definedbiological samples and for the identification of the papillomavirusesaccording to the types or subtypes to which they belong. The conditionsunder which these products of gene expression may be obtained will beillustrated in the remainder of this description, particularly withregard to the expression of the L2 sequence of papillomavirus HPV1a, itbeing understood that similar techniques may be used to induce theexpression of the genetic sequences corresponding to L2 (or E6, E7 orL1) from other types of papillomavirus. In the discussion which follows,particular reference will be made to the sequences or genescorresponding to L2, it being nonetheless understood that theinformation furnished relating to the products of expression of the L2genes may, if required, be applied to the products of expression of theother gene referred to above. Nonetheless, particular interest attachesto the products of expression of the L2 sequences, in that they maythemselves be used for the in vivo production of antibodies capable ofrecognizing the products of expression of the L2 gene in biologicalsamples infected with the corresponding papillomavirus but not with apapillomavirus of a different type, and more particularly when thepreparations of this type have been fixed beforehand. The new procedureprovided by the invention and which will be discussed later thusprovides distinct agents which make it possible to determine the typesof papillomavirus present in the lesion under study so that theirseverity can be assessed and the appropriate treatment chosen. Theantibodies produced against the different types of products expressed bythe L2 genes can be grouped in mixtures corresponding to those whichwere identified earlier in the context of the hybridization probes.

Consequently, the invention also provides "kits" containing severaldistinct antibodies which can be used in a series of tests to detect,and if necessary, identify or classify, the newly isolatedpapillomaviruses. For example, a kit in accordance with the inventioncontains a number of reagents constituted by distinct antibodies ormixtures of antibodies, for example reagents comprising antibodiesformed against the products expressed by the L2 genes ofpapillomaviruses constituted in the following groups:

1) at least HPV2d,

2) at least one of the HPVs can be 10b, 28 and 29,

3) at least one of the HPVs 17 and 24,

4) at least one of the HPVs 14, 15, 17, 19, 20, 21, 22, 23 and IP4,

5) at least one of the HPVs 15 and 17,

6) HPV 24,

7) at least one of the HPVs 14, 32 and IP4,

8) HPV 31,

9) HPV 32,

10) at least one of the HPVs 16, 18 and IP2,

it being understood that the antibodies of the ten groups are chosen insuch a manner as to be different from each other under all circumstancesshould each of the ten groups be reduced to only one of the antibodiesof which they are composed.

The antibodies formed against the products expressed by the L2 genes (oragainst recombinant proteins containing these products of expressionfused with an additional polypeptide, in particular a stabilizingpolypeptide which does not lead to the modification of the immunogenicproperties of the product expressed by the L2 genes) are used for thedirect detection of virus in histopathological sections derived from thelesions induced by the papillomaviruses in the subject infected by them.Advantageously, the detection is carried out on preparations fixedbeforehand under dissociating conditions, for example, in the CARNOYmixture already cited (also described in the monograph by L. LISON,entitled "animal histochemistry and cytochemistry").

The anti-L2 antibodies, fixed if possible, may be recognized by otherantibodies formed against the first antibodies and to these secondantibodies appropriate markers, preferably non-radioactive ones, may beattached. For example, these markers may be enzymes or fluorescentprobes.

Quite naturally, the invention also relates to the polypeptidesthemselves, which are the products of expression of the L2 genes of thepapillomaviruses. These products of gene expression have already beenreferred to earlier as "L2 proteins". The invention also relates to thepolypeptides in which this L2 protein is fused to other polypeptidesequences provided that these latter do not cause any criticalmodification of the immunogenic properties of the L2 protein. Thepresence of these other polypeptide fragments can arise notably from themethod used to produce these hybrid polypeptides, particularly when theyhave been prepared by procedures involving the techniques of geneticengineering. Advantageously, the invention relates to hybridpolypeptides containing a sequence derived from beta-galactosidase. Suchproducts may be obtained notably by transformation of E. coli withappropriate vectors (phages or plasmids) modified by all or part of thelactose operon and containing, in addition, downstream of the lactoseoperon promoter (or any other appropriate promoter, for example of phagelambda) the nucleotide sequence derived from the L2 gene of a specifictype of papillomavirus. It is an advantage to be able to make use ofplasmids or phages of this type containing a part at least of thebeta-galactosidase gene of the lactose operon.

The invention also relates to groups of distinct polypeptides, eachgroup of polypeptides corresponding to only a part of the complete L2proteins referred to above, it being nonetheless understood that thesedifferent groups of polypeptides each contains the antigenic sitescharacteristic of the L2 proteins of the type in question.

When they have been purified, the polypeptides according to theinvention may also be employed in methods used to purify theircorresponding antibodies, particularly from the sera of animals whichhave been immunized with these polypeptides. In particular, thesepolypeptides may be bound to affinity columns. The procedures for thepurification of the antibodies then consist of allowing the serumcontaining them to pass through an affinity column to which theabove-mentioned polypeptides are attached. The antibodies selectivelybound to these columns can then be recovered by dissociation of theantibody-antigen complexes by means of an appropriate buffer ofsufficient ionic strength, for example, a solution of a salt such asammonium acetate. Acidic solutions may also be used.

Finally, the invention relates to mixtures involving these antigens (orgroups of antigens) and antibodies (or groups of antibodies).

In particular, the invention relates to groups containing one or,preferably a "cocktail" of antibodies derived from sets ofpapillomaviruses, all of which are often reputed to be present in agiven type of disease. These cocktails (containing these antibodies orgroups of antibodies: mixtures of sera or preparation of purifiedantibodies in association with an appropriate pharmaceutical excipient)are then available for use by administration, in particular by theparenteral route, to the patient concerned for the treatment of a givendisease, as soon as the latter has been diagnosed clinically. Thediagnosis results from an in vitro diagnostic test performed on acytological or histological sample taken from the patient or from an invivo diagnostic test which has shown that the infectious papillomavirusbelongs to a type similar to that of one of the set of papillomavirusesreferred to above. These sera are then able to cause a regression of theinfections induced by the corresponding types or subtypes ofpapillomaviruses.

Finally, the invention relates to the corresponding preparations ofvaccines containing one or, preferably, several L2 proteins incombination with an acceptable pharmaceutical excipient adapted to thechosen route of administration, in particular to the parenteral routewhich is used in order to protect the patients from the high risk ofbeing infected with the corresponding disease.

Finally, reference is made to articles, together with the appropriateliterature references, which supplement the description of the state ofthe prior art to the extent that that mav be necessary or prove usefulfor the reader's complete understanding of the text. In this sense, thecontent of these articles must thus be considered to constitute part ofthe description.

A preferred methodology for the production of an L2 protein (or afragment of this protein) which makes use of a fragment of the L2 geneof a given papillomavirus in the proper reading frame will beillustrated in connection with the description of the construction of avector incorporating such fragments in the proper reading frame,followed by a description of the conditions under which the expressionof the L2 protein or the polypeptide fragment can be induced in E. coli.A procedure for the purification of the protein or the polypeptide aswell as its use in the production of sera containing antibodies directedagainst the protein or polypeptide will also be described. It will beclear to the specialist that the L2 fragment used in the proper readingframe will need to be fused into the vector used each time in a mannersuch as to preserve the open reading frame. If possible, it is useful tocarry out fusion with a gene coding for a protein which guarantees thestability or facilitates the subsequent purification of the hybridprotein thus formed.

The steps of the production procedure are illustrated in FIG. 11 of theappendices.

MATERIALS AND METHODS

1) TREATMENT OF THE DNAS

For procedures relating to DNA recombination, the preparation of theplasmids. and the DNA fragments, the formation of non-sticky ends, thedigestion with the enzyme Bal 31 and finally the transfection of thecells, recourse was had to the methods of Maniatis, T. et al. (1982),described in the chapter entitled "Molecular cloning" in the monographentitled "A Laboratory Manual", Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., USA.

2) BACTERIA, PLASMIDS AND VIRUSES.

The plasmid DNAs were obtained from pHPVI.a (Danos et al., 1982 andFrench patent 82 05887 of Apr. 5, 1982) contained in E. coli. (strainC600) pCQV2 expressed in the strain RRI (Queen, 1983) and pMC1403expressed in strain MC1000 (Casadaban, 1983).

The intermediate plasmids constructed and the plasmid pHPL2 were passedseveral times in cultures of E. coli MM 294 and the plasmidspHPL2-beta-galactosidase in the strain MC1000 deficient inbeta-galactosidase. The Lac+strain were detected as red colonies onplaques of Mc Conkey agar containing lactose (Silhavy et al., 1984, in"Experiments with gene fusions": Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.).

The plasmids pHPL2 and pHPL2-beta-gal were then transfected into a lon⁻strain deficient in protease, CAG1139 (Grossman et al., 1983, Cell 32,151-159).

3) PREPARATION OF BACTERIAL LYSATES AND THEIR ANALYSIS ON POLYACRYLAMIDEGEL IN THE PRESENCE OF SODIUM DODECYLSULFATE (SDS-PAGE).

After being cultured overnight, CAG 1139 cells containing pHPL2 orpHPL2-beta-gal 116 were diluted 5 to 20 fold with LB medium. They werethen grown at 30° C. until the optical density 600 had attained 0.5-0.9.

The PR-lambda promoter was then derepressed by growing the cells at 41°C. for 90 minutes. The cultures were then recentrifuged and the pelletscollected and suspended in a medium of 62 mM Tris, pH 6.8, containing 2%SDS, 26% glycerol, 2M 2-mercapto-ethanol and 0.03% bromophenol blue. Alysate of CAG 1139 cells not containing plasmid was used as control.

After being heated at 100° C. for 10 minutes, the samples were subjectedto electrophoresels in a 10% SDS--polyacrylamide gel according to thetechnique of Laemli (1970), Nature, 227, 680-684. BRL prestainedproteins of known molecular weights were also subjected toelectrophoresis.

The protein bands were visualized by treatment with a solution of thedye known as "Amido black" and transferred to a sheet of nitrocellulosefor analysis by the method known as "Western Blot".

4) ANALYSIS

The analysis by "Western Blot" was carried out as described by Rosettoet al. (1984) J. Gen. Virol. 65, 1319-1324, by using papillomaviruspolyclonal antibodies (Orth et al. (1978), Virology 91, 243-255 andRosetto et al., already cited) and rabbit and guinea-pig anti-IgGimmunoglobulins conjugated to horseradish peroxidase.

5) ANALYSIS OF ANTIBODIES

Use was made of the techniques of immunodiffusion, immunofluorescenceand immunoperoxidase previously described (Orth et al., 1984, andRosetto et al., 1984) in conjunction with purified HPV1.a viralparticles or sections of the lesions induced by distinct types ofpapillomaviruses.

6) PURIFICATION OF L2-BETA-GALACTOSIDASE FUSION PROTEINS

A culture of CAG 1139 containing the plasmid pHPL2 beta-galactosidase116 was diluted 200 fold with LB medium and the cells were then grownuntil they had attained O.D. of 0.32. The culture was then broughtrapidly to 41° C. and maintained at the higher temperature for 90minutes.

The pellet of cells was resuspended in 20 mM Tris buffer, pH 7.5,containing 10 mM Mg Cl₂ and the cells were disrupted by sonication.

The fusion protein thus released into the medium was purified byaffinity chromatography on a column ofp-aminophenyl-B-D-thiogalactosidasi (TPEG)-SEPHAROSE as described byUllmann (1984). Gene 29, 27-31.

The fusion protein, purified as indicated above, was used to immunisefemale Harley guinea-pigs. The animals received three sub-cutaneousinjections, at distinct sites, of about 150 micrograms of protein in thepresence of complete Freund adjuvant (DIFCO) at intervals of two tothree weeks. The serum was collected one week after the third injection.

RESULTS

CONSTRUCTION OF PLASMIDS CAPABLE OF EXPRESSING THE L2 OPEN READING FRAMEIN ESCHERICHIA COLI

The constructs are presented in FIG. 11 The L2 open reading frame wascloned with a view to its expression in Escherichia Coli (E. coli). ThepHPV1.a plasmid contains the entire genome of HPV1.a cloned at the BamHIsite of the plasmid pBR322 by Danos et al. (EMBO J.1, p. 231-236, 1982).For the purpose of isolating open reading frames of the late region ofthe viral genome, a HpaII-HindIII fragment of a pHPV1.a plasmid- wassubcloned into a pBR322 plasmid. The plasmid obtained was called pHPL9.This plasmid was shortened by removing a non-essential PVu II-BamHIfragment in the pBR322. From the resulting plasmid pHPL 9.3, aXmnI-XhoII fragment containing 1526 base pairs (b.p.) of the openreading frame minus the 318 base pairs of the 5' terminals was isolated.

The stop codon of the L2 open reading frame is conserved.

The L2 fragment obtained was then inserted into the expression vectorpCQV2 (Queen 1983) in place of a non-essential PvuII-BamHI fragment. TheL2 open reading frame is thus found directly adjacent to the ATG stopcodon and to the SD sequence (Shine and Dalgarno, Proc. Natl. Acad. Sci.

USA 71, p. 1342-1346, 1974) of the cro gene of the phage lambda andunder the control of the PR lambda promoter. The promoter is regulatedby the temperature-sensitive repressor CI857, which thus providescontrol of the production of the product expressed by the L2 gene. Inthis way, the plasmid pHPL2 was obtained in which the 1206 base pairs ofthe C terminal part of the L2 open reading frame are in phase with theATG start codon of the cro gene of the lambda phage. The presence of aXhoII restriction site at the BamHI/Bgl II junction provided proof thatthe open reading frame had been maintained.

With the aim of stabilizing the L2 protein synthesized in E. coli and atthe same time of making available a convenient method for itspurification, it was also decided to allow it to be expressed as ahybrid protein fused with beta-galactosidase of E. coli.

In order to achieve this, the L2 terminal codon was eliminated afterlinearization of the pHPL2 plasmid at the BstEII site, followed bydigestion with the enzyme Bal31.

This DNA, which contains the L2 gene and the sequences supplying thesignals for transcription and translation, was shortened by digestionwith PstI, before being inserted into the plasmid pMC1403 (Casadaban,Methods in Enzymology 100, p. 293-308, 1983) in place of the SmaI-PstI.

PMC1403 contains the lac operon without its promoter and the first 22base pairs of the open reading frame for the gene of beta-galactosidase.The recombinant was transfected into MC 1000, lac⁻ cells. The productionof beta-galactosidase in the clones obtained at high temperatureprovided a means to select on McConkey lactose agar plates (Silhavez etal., Cold Spring Harbor Laboratory, N.Y. 1984) the pHPL2-beta-galplasmids in which the genes for L2 and beta-galactosidase are both inthe open reading frame.

The pHPL2-beta-gal 116 clone, selected by such a procedure, was studiedlater.

The sequencing of the DNA (result not shown here) indicated that onlythe last two C terminal amino acids of the L2 open reading frame hadbeen lost in the course of digestion with Bal 31 and that the productexpressed by the L2 open reading frame was linked through a prolineresidue to the ninth amino acid of beta-galactosidase.

PRODUCTION OF PROTEINS RELATED TO HPV1.a BY CELLS TRANSFECTED WITH pHPL2AND pHPL2-BETA-GAL 116

The plasmid pHPL2 has the capacity to code for an L2 protein of about51.2 Kd and the pHPL2-beta-gal plasmid for a hybrid protein of about 167Kd.

In order to study the production of proteins by these plasmids, thelatter were first transfected into a strain of E. coli CAG 1139 lon⁻,which is protease deficient. The cells were then cultivated at 30° C.before being brought to 41° C. in order to induce the production of theproteins under the control of the PR lambda promoter. The bacteriallysates obtained were electrophoresed on an SDS-polyacrylamide gel.

The proteins related to this virus HPV1.a were detected by analysis bymeans of the technique called "Western Blot" using anti-HPV1.a virusantibodies. For the cells grown at 30° C., the analytical results werethe same, whether or not they contained a plasmid. However, in the caseof the cells induced at 41° C., specific bands of protein weredemonstrated in the transfected cells: among the proteins isolated fromthe cells transfected with the recombinant pHPL2-beta-gal 116, it aspossible to isolate a main band having the expected molecular weight ofapproximately 167 Kd, corresponding to an L2 beta-galactosidase(L2-beta-gal) fusion protein, and several minor bands of moleculareights of about 58 Kd, probably resulting from proteolytic degradation.

The plasmid. pHPL2 gave rise to a protein band of about 72 Kd. Thismolecular weight is higher than the expected one of 51.2 Kd. Thesignificance of this es remains to be elucidated.

In order,then,to be able to study the antigenicity of the proteinproduced in E. coli, the L2-beta-gal product was purified from thebacteria lysate of a culture induced by heat,by means of affinitychromatography oon a column of TPEG-SEPHAROSE (Ullman, Gene 29, p.27-31, 1984). The proteins eluted from the column were analysed from anSDS-polyacrylamide gel. Staining with "Amido-Black" revealed threeprinciple protein bands, one with a high molecular weight probablycorresponding to the hybrid protein L2-beta-gal, the second migratingtogether with purified beta-galactosidase and a third with a molecularweight of about 60 Kd.

This protein with the low molecular weight may be a contaminant.Analysis by the technique of "Western Blot" with a specific antibody ofthe HPV1.a type reveals a main band of high molecular weightcorresponding to the product expressed by the fused L2-beta-gal gene andseveral minor bands (FIG. 3b).

Immunogenicity of the L2-beta-gal Product

In order to test the immunogenicity of the fusion protein, the producteluted was injected into two guinea-pigs. The sera were collected afterthe third injection. By means of a "Western Blot" analysis, the seraobtained reacted with bands migrating as the L2-beta-gal fusion proteinor as the L2 product in the bacterial lysates derived from culturescontaining the plasmids pHPL2-beta-gal 116 and pHPL2, respectively, andwhich had been induced by a temperature shift.

In the two types of cell lysate the sera recognized proteins (about 60and 55 Kd) which were also found in the control lysate CAG 1139. Atleast one of these proteins appeared to correspond to the proteincopurified with the L2-beta-gal fausion protein and beta-galactosidase.

The sera recognized proteins of about 80 Kd in a dissociated HPV1.aviral preparation (treated with a detergent) and in an extract of a wartinduced by HPV1.a.

In an immunodiffusion test, the guinea-pig sera obtained did notprecipitate the intact HPV1.a virus particles used as antigen, a findingwhich indicates once again that the viral antigens recognized by theserum in the immune transfer technique are not available at the surfaceof the virion or that they are not precipitated by the sera.

The sera induced by the products expressed by the L2 gene did not reactwith frozen sections of HPV1.a warts which are known to preserve thenative conformational structure of the virions and of the viralpolypeptides. This result testifies once again to the fact that theantibodies are not directed against a conformational antigen.

The sera reacted only slighly (at a dilution of 1/50) with sections ofwarts infected with HPV1.a which had been fixed in BOUIN medium. Thisresult confirms the specific character of the type of antigens involvedas opposed to groups of antigens which should lead to strongerantibody-antigen reactions with sections infected by a virus and fixedin BOUIN medium.

On the other hand, the observations made on sections fixed in CARNOYmedium presented evidence of positive reactions both with the typeantigens and the group antigens. In particular, it was observed that theguinea-pig sera gave very strong antibody-antigen reactions (even at adilution of 1/1000) when the section contained a virus of the same typeas that which had furnished the antigen which had served to induce theantibodies used in the assay. On the other hand, no immunologicalcross-reaction was observed when the serum under study was placed incontact with sections containing a virus belonging to a different type.

It needs also to be pointed out that the type antigens considered arenot necessarily always the same as the types such as those defined abovewith regard to the mutual capacities for hybridization of genomes of twodistinct papillomaviruses.

In particular, it is important to bear in mind the distinction betweentwo kinds of antigens in papillomaviruses:

the type antigens, characterized by the absence of an antigeniccross-reaction between the different kinds of papillomavirus when usingantibodies obtained by injection of whole virions,

and the group antigens essentially masked in the virion, and thepresence of which is demonstrated by antibodies after injection ofdissociated viral particles.

The papillomaviruses belonging to different types in the assays ofantigen-antibody reactions are those for which the L2 coding sequences,from which their respective homologous regions have preferably beenremoved, code for polypeptides which do not give rise toantibody-antigen cross-reactions with their respective antibodies.

Preferred types of antigens are those which are coded by the openreading frames of the L2 gene lacking the N terminal region, comprisingin particular up to 1/4 of the length of the L2 regions in question.

Finally, the invention also relates to a test for the detection of thosepolypeptides coded by the L2 regions which do not give cross-reactions,in viruses belonging to different types. These polypeptides may bedefined as those, the antibodies of which react efficaciously withviruses contained in cytological or histological sections fixed inCarnoy medium, but which react poorly, if at all, with the same sectionswhen these latter are fixed in Bouin medium.

The invention also relates to a procedure for the classification of newpapillomaviruses relative to known papillomaviruses.

The procedure is characterized in that, after hybridization of itsgenome under non-stringent conditions with the L2 region of one orseveral genomes of known papillomaviruses or, if the occasion arises,sequencing of its genome, and the consequent identification of theopen-reading frame of the L2 gene, a recombinant is produced between afragment consisting of all or part of this L2 gene which has beenexcised from said genome beforehand and an appropriate vector. Then theexpression of this fragment is induced in a host cell or appropriatemicroorganisms, in particular E. coli. If desired, antibodies areproduced in vivo against the products of expression which have beenobtained and assays of antibody-antigen cross-reactions are carried outbetween either the products expressed by the L2 genes of the newlyisolated papillomavirus or the corresponding antibodies, on the onehand, and either the antibodies or the antigens corresponding topapillomaviruses of known types, on the other. The type of the newlyisolated papillomavirus can be distinguished from or identified withthose of known papillomaviruses, depending on whether theantigen-antibody cross-reactions give negative or positive results.

Finally, the invention also relates to the diagnostic procedure whichincludes the identification of the type of infectious virus which may bepresent in a patient and consists of allowing antigens previouslyobtained from viruses belonging to different types to react with abiological sample, in particular a serum sample, taken from the patienton whom the test is being conducted.

The infectious virus will be assumed to belong to a specific type whenan antibody-antigen reaction is observed between a serum sample and anantigen derived from a papillomavirus belonging to the same type. Thesediagnostic assays may be carried out, for example, by using the ELISAmethod.

More particularly, the invention relates to a procedure for theidentification of an infectious papillomavirus in a sample taken from apatient such as tissue or fluid, for example serum, with a given type ofpapillomavirus. This procedure is characterized by placing thisbiological sample in contact with antibodies which had previously beenformed against the product expressed by a DNA containing at least a partof the L2 region of the genome of a virus belonging to this type, thisadmixing being performed under conditions and for a time which allowedan immunological reaction to take place. The detection of the formationof an antibody-antigen complex then provides evidence for the presenceof a papillomavirus of a type identical or related to that which hadgiven rise to the test antibodies.

Preferably, the DNA containing the L2 region or a part of it was clonedin a competent host cell such as a bacterium, for example, E. coli.

Advantageously, the part of the L2 region utilized corresponds to theopen reading frame for the L2 gene lacking the N-terminal region, whichis not characteristic of the type. Preferably, this N-terminal regioncorresponds to the first quarter of the reading frame.

More particularly, the invention relates to a detection procedure ofthis type in which the DNA containing the above-mentioned part of the L2region (or the entire L2 region) is a DNA hybrid formed from a nucleicacid coding for a protein normally expressed in the chosen competenthost cell and in which the above-mentioned L2 region has beenincorporated beforehand, in particular by in vitro recombination.

For example, the protein normally expressed in the host cell correspondsto all or part of beta-galactosidase, when the host cell is E. coli.

The detection procedure according to the invention is applicable to allhistological sections which have been fixed, preferably in Carnoymixture,and also directly to a serum sample.

The procedure according to the invention also comprises the addition ofthe biological sample under the above-mentioned conditions to"cocktails" of antibodies previously formed against products expressedby the L2 genes--or part of them--derived from several viruses of thesame types or of related types, in particular of viruses classified inthe same manner as that indicated above with respect to the DNA probes.

The use of these "cocktails" of antibodies under conditions similar tothose which were mentioned in connection with the DNA probes thus enablecorrelations to be made between the group to which a givenpapillomavirus belongs and the disease from which the patient understudy is suffering or to which he is potentially exposed.

Finally, the invention relates to a procedure for the preparation ofeach of the above mentioned hybrid polypeptides and antibodiescorresponding to them.

This procedure comprises:

the incorporation, in vitro in particular, of the whole or part of theL2 region of the genome of the papillomavirus concerned into anappropriate vector;

the transformation of a competent host cell with the vector thusmodified, i.e. a host capable of expressing the whole or part of theabove-mentioned L2 region;

the recovery and,preferably, the separation of the polypeptide resultingfrom the expression of the whole or part of the L2 region, thispolypeptide being capable of inducing in vivo the production ofantibodies capable of detecting papillomavirus proteins under theconditions set out above.

The invention also concerns the procedure for the production of the saidantibodies, characterized by the immunization of an animal, a rabbit forexample, with the above-mentioned polypeptides, and the recovery of theantibodies formed.

Finally, the invention may be extended to include the classification ofeither the hybrid peptides or the antibodies, obtained from variouspapillomaviruses according to the different types to which they prove tobelong.

REFERENCES

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We claim:
 1. A kit comprising:a) at least one antibody that specificallybinds to an L2 protein of a papillomavirus, wherein said antibody:bindsan L2 protein of said papillomavirus, which L2 protein lacks theN-terminal homologous region comprised in the N-terminal 25% of theamino acid residues of the entire papillonxavirus L2 protein; and, doesnot cross react with L2 proteins encoded by DNA sequences ofpapillomaviruses which exhibit cross-hybridization of less than 50% withsaid papillornavirus DNA sequences under stringent conditions, andwherein said papiliornavirus is selected from HPV 1a, HPV 2d; HPV 10a,HPV 10b, HPV 14a, HPV 14b, HPV 15, HPV 17a, HPV 17b, HPV 19, HPV 20, HPV21, HPV 22, HPV 23, HPV 24, HPV 28, HPV 29, HPV 31; HPV 32, HPV IP2 andHPV IP4; and, (b) a label for detecting an antibody-antigen reaction. 2.The kit of claim 1, wherein said papillomavirus is papillomavirus HPV1a.
 3. The kit of claim 1, wherein said L2 protein of saidpapillomavirus is the expression product in E. coli host cells of arecombinant nucleic acid encoding said L2 protein.